Cell migration is central to numerous biological and pathological processes, including embryogenesis, the inflammatory response, tissue repair and regeneration, cancer, arthritis, atherosclerosis, osteoporosis, and congenital developmental brain defects. Yet, despite its importance, migration is currently not well understood on a molecular level. Migration may be viewed as a multi-step cycle, which includes extension of a protrusion, usually induced by an exogenous agent, formation and disassembly of attachments (adhesions) near the leading edge of the protrusion, translocation of the cell body forward, and retraction of the cell rear. The coordinated regulation of these processes is dependent on remodeling of the actin cytoskeleton. Asef2 is a recently identified protein that has been shown to activate Rac, which is a key regulator of the actin cytoskeleton; however, the function of Asef2 in modulating cell migration is currently not well understood. The goal of this proposal is to investigate the role of Asef2 in controlling cell migration through its ability to regulate underlying processes. Aim 1 will probe the function of Asef2 and Rac in migration by altering protein expression and by generating mutants that affect their activity. In this aim, migration wil be analyzed on two dimensional (2D) type I collagen, which is an extracellular matrix protein that promotes cell attachment, and in three dimensional (3D) type I collagen gels. Since tissues and organs are three dimensional, 3D matrices will be used to recapitulate the in vivo environment of cells. Aim 2 will explore the mechanism by which Asef2 regulates migration by testing the hypothesis that Asef2 controls migration through Rac and the actin motor protein myosin II. In support of this hypothesis, preliminary results show that Asef2 increases contractility, and myosin II is known to be important for actomyosin contractility. In this aim, expression constructs, mutants, and inhibitors will be employed to determine if Asef2 regulates migration through myosin II. In Aim 3, the role of Asef2 in modulating adhesion assembly and disassembly (adhesion turnover) will be explored using total internal reflection fluorescence microscopy, an advanced microscopy technique. Moreover, an approach similar to that outlined in Aim 2 will be used to determine if Asef2 regulates adhesion dynamics through Rac and myosin II. The proposed studies will significantly enhance our understanding of the molecular factors that regulate cell migration. This could lead to new therapeutic approaches for the various pathological disorders, which can be attributed to uncontrolled cell migration.